The DDT1 cell line has been reported to be an androgen-responsive cell line. Norris et al. "Androgen Receptors in a Syrian Hamster Ductus Deferens Tumour Cell Line," Nature 248:422-424 (1974). Syms et al., "Glucocorticoid Effects on Growth, and Androgen Receptor Concentrations on DDT.sub.1 MF-2 Cell Lines", J. Steroid Biochem., 28(2):109-116 (1987) report that the DDT.sub.1 -MF-2 smooth muscle tumor cell line contains receptors for and is differentially sensitive to androgens and glucocorticoids. They report that androgens stimulate growth, and glucocorticoids inhibit growth of the cell line.
Harris et al., "Androgens and Glucocorticoids Modulate Heparin-Binding Growth Factor I mRNA Accumulation in DDT1 Cells as Analyzed by in situ Hybridization", Mol. Endo. 3(11):1839-1844 (1989), describe the ductus deferens smooth muscle tumor cell line (DDT.sub.1 -MF-2) as steroid-sensitive. They report that treatment with 10 nM testosterone accelerated the growth of DDT1 cells in the absence of serum, and that glucocorticoids inhibit growth. They further identify the DDT.sub.1 -MF-2 cell line as a useful model for steroid responsive tumor growth in vitro.
There has been a need for tissue culture models that mimic the androgen responsiveness observed in androgen sensitive diseases, particularly that observed in human prostatic carcinomas. The present invention provides for an assay to study compounds as potential anti-androgens in living cells.
Lippman et al. in "The Effects of Androgens and Antiandrogens on Hormone-response Human Breast Cancer in Long-Term Tissue Culture" Cancer Research 30: 4610-4618 (1976), describe the MCF-7 androgen-responsive cell line. Unlike the cell line of the present invention, the MCF-7 cell line cannot be grown in synthetic media.
Similarly, Marugo et al. in "Effects of Dihydrotestosterone and Hydroxyflutamide on Androgen Receptors in Cultured Human Breast Cancer Cells (EVSA-T)" J. Steroid Biochem. Mole. Biol. 42(5):547-554 (1992), describe another androgen responsive cell line. However, like the cells of the MCF-7 cell line, these cells cannot be grown in synthetic media.
The human androgen receptor has been isolated and characterized.
Tilley et al., "Characterization and Expression of a cDNA Encoding the Human Androgen Receptor", Proc. Nat'l. Acad. Sci. USA, 80:327-331 (1989) report the isolation, characterization and expression of the cDNA encoding the human androgen receptor, which predicts a protein of 917 amino acids and a molecular weight of 98918.
Rasmusson et al., "Therapeutic Control of Androgen Action", Ann. Rep. Med. Chem. 29:225-234 (1994) present a review of the androgen receptor, chemical antagonists of the androgen receptor, and means of controlling androgen biosynthesis.
Compounds identified as anti-androgens by the assay of the present invention are especially useful in the prevention and treatment of prostatic carcinoma, and they may also be useful in the treatment and prevention of other hyperandrogenic diseases such as acne vulgaris, seborrhea, female hirsutism, androgenetic alopecia, also called androgenic alopecia, which includes male and female pattern baldness, and benign prostatic hyperplasia.
Benign prostatic hyperplasia (BPH) and prostatic carcinoma are among the most common afflictions of aging men.
Benign prostatic hyperplasia is often treated surgically with a procedure known as transurethral resection of the prostate (TURP). Other surgical procedures performed to release the obstruction of urine include incision or stents. Castration has also resulted in regression of prostatic enlargement. Drug therapy for BPH has included alpha-1 blockers which treat the symptoms of the disease by alleviating obstructive symptoms, but do not affect the underlying cause of the disease, the enlarged prostate gland. Representative alpha-1 blockers used in the treatment of BPH include: prazosin, terazosin, doxazosin, tamsulosin and alfuzosin. These drugs relax prostatic smooth muscle tone, decreasing intraurethral pressure without affecting bladder pressure. Common side effects of these agents are dizziness, headache and fatigue.
Finasteride (17.beta.-(N-tert-butylcarbamoyl)-4-aza-5.alpha.-androst-1-ene-3-one), which is marketed by Merck & Co., Inc., under the tradename PRQSCAR.RTM., is an inhibitor of testosterone 5.alpha.-reductase currently marketed for the treatment of benign prostatic hyperplasia. A principal mediator of androgenic activity in the prostate is 5.alpha.-dihydrotestosterone ("DHT"), formed locally in the prostate by the action of testosterone-5.alpha.-reductase. Inhibitors of testosterone-5.alpha.-reductase inhibit the conversion of testosterone (T) to DHT and serve to prevent or lessen symptoms of hyperandrogenic stimulation in the prostate. See especially U.S. Pat. No. 4,377,584 assigned to Merck & Co., Inc., issued Mar. 22, 1983. The utility of finasteride in the treatment of prostatic carcinoma is also disclosed in the following documents: EP 0 285,382, published 5 Oct. 1988; EP 0 285 383, published 5 Oct. 1988; Canadian Patent no. 1,302,277; and Canadian Patent no. 1,302,276.
Both prostatic carcinoma and BPH have been treated with antiandrogens. Nonsteroidal antiandrogens such as flutamide and Casodex compete with DHT for androgen receptor sites in the prostate cells. These non-steroidal antiandrogens do not substantially change sexual potency and libido as the gonadotrophin releasing hormone agonists and progestogens do; however, these nonsteroidal antiandrogens often exhibit the undesirable tendency to feminize the male host (gynaecomastia) or initiate feed-back effects which would cause hyperstimulation of the testes.
Gonadotrophin-releasing hormone (GnRH) agonists such as nafarelin, buserelin, goserelin and leuprorelin all reduce the release of leutinizing hormone (LH) by desensitizing the GnRH receptors in the anterior pituitary gland. GnRH agonists are able to reduce the production of testosterone, induce shrinkage of prostate volume and reduce the severity of urinary symptoms of BPH. Unfortunately, these drugs have adverse effects such as impotence and flushing, which discourage a majority of patients from continuing with the drugs. These androgen-suppressing agents are thus of inconsequential significance in BPH treatment, but are of major importance in the treatment of patients with advanced prostatic cancer.
Progestogens, such as megestrol acetate, hydroxyprogesterone and medrogestone depress testosterone by inhibiting LH release and blocking androgen receptors, causing a reduction in prostatic volume. Adverse effects such as decreased libido and impotence have kept progestogens from common use in BPH treatment.
Thus, there still remains a need for additional therapies for BPH and prostatic carcinoma for individuals who cannot tolerate the side effects and/or do not experience adequate relief from presently available therapies.
There also remains a need for a compound for the treatment of diseases of the prostate that is a non-steroidal compound having different pharmacological properties from steroids.
Hori et al. "WB2838 3-Chloro-4-(2-amino-3-chlorophenyl)-pyrrole!: Non-steroidal androgen-receptor antagonist produced by A Pseduomonas" J. Antibiotics 46(9):1327-1333 (1993) describe the nonsteroidal androgen receptor antagonist labeled WB2838: ##STR3##
N-(4-chlorophenyl)-(Z,Z)-2,3-bis(cyclopropylmethylene) cyclopentane carboxamide is described by T. Wong "Synthesis nad Some Related Studies of Alkyl 2,3-bis(alkylidene)cyclopentance carboxylates", Ph.D. thesis, University of British Columbia, December, 1993. No use for this compound is described in the thesis.
Steroid hormones are involved in numerous aspects of cell growth and differentiation. In an effort to influence these processes chemists have developed analogs usually based on the four ring steroid nucleus. Nonsteroidal compounds interacting with steroid receptors are more rare and exhibit different pharmacological properties from their steroidal counterparts. We describe the use of androgen receptor binding assays and androgen-dependent DDT1 cells to identify and characterize novel antiandrogens, of particular interest are nonsteroidal antiandrogens.